The field of the invention is expression and purification of cloned human alpha-fetoprotein.
Alpha-fetoprotein (AFP) is a serum protein normally found at significant levels only in fetal blood. In adult blood increased alpha-fetoprotein levels are associated with liver regeneration and certain carcinomas.
The specific function of alpha-fetoprotein is not known. Suggested roles for the protein include: fetal albumin; protection from maternal immune attack; and protection from maternal estrogen.
Morinaga et al. (Proc. Natl. Acad. Sci. USA 80:4604, 1983) report the cloning of human AFP.
Innis et al. (Arch. Biochem. Biophys. 195:128, 1979) report the cloning of an approximately 950 base-pair fragment of human AFP into E. coli plasmid pBR322.
Nishi et al. (J. Biochem. 104:968, 1988) report the expression of rat AFP in E. coli and Saccharomyces cerevisiae. Nishi et al. also report that, in an estradiol-binding assay, yeast-produced rat rAFP is as active a authentic AFP, while bacterial-produced rat rAFP is essentially inactive. Further, when characterized by radioimmunoassay or an Ouchterlony double immunodiffusion assay, yeast-produced rat rAFP bears a closer resemblance to authentic rat AFP than does bacterial-produced rat rAFP. Nishi et al. state that:
"In the Ouchterlony double immunodiffusion test, authentic and yeast rAFP formed a completely fused precipitin line with antibody to rat AFP while E. coli rAFP showed a reaction of partial identity in a similar test . . . . It is likely that the functionally active yeast rAFP in this study had the correct pairs of disulfide bridges. On the other hand the E. coli rAFP probably failed to form them". PA1 a) providing a transformed insect cell (e.g., Spodoptera frugiperda) including a recombinant DNA molecule encoding the human alpha-fetoprotein or fragment or analog thereof operably linked to an expression control element which directs the expression of the human alpha-fetoprotein or fragment or analog thereof; PA1 b) culturing the transformed cell; and PA1 c) recovering the biologically active human alpha-fetoprotein or fragment or analog thereof.
Yamamoto et al. (Life Sciences 46:1679, 1990) report the expression of human AFP in yeast and report that the rAFP so produced was "indistinguishable immunologically from authentic AFP."
Giuliani et al. (Protein Engineering 2:605, 1989) report the expression of a portion of human AFP (amino acids 38 to 119) in E. coli.
Japanese Patent Application 88158596 reports a method for preparing recombinant human domain I AFP in E. coli.